The rapid degradation of specific regulatory proteins underlies a wide range of dynamic cellular and developmental processes. Such short-lived proteins include products of the proto-oncogenes, cell cycle regulators and proteins controlling development and differentiation. A number of distinct proteolytic pathways operate in eukaryotic cells, some of which depend on the highly conserved polypeptide ubiquitin. The objective of the research described in this application is to gain an understanding of the different signals within short-lived regulatory proteins that cause them to be rapidly destroyed in vivo and to characterize, by molecular genetic approaches, the molecular machinery involved in ubiquitin-dependent and ubiquitin-independent degradation. The following specific aims will exploit a recently developed model system that is based on the short-lived MATalpha2 transcriptional repressor of the yeast Saccharomyces cerevisiae. Given the extraordinary conservation, between yeast and higher eukaryotes, of fundamental regulatory systems in general (e.g., the cell cycle) and of the ubiquitin system in particular, the results of the work outlined in this proposal should also provide important insights into regulatory protein degradation and its role in cellular metabolism, growth, and differentiation in higher eukaryotes. 1. Identification and characterization of the molecular determinants in the alpha2 repressor that render it short-lived in vivo using in vitro mutagenesis methods. 2. Genetic analysis of the enzymatic pathways responsible for the ubiquitination and/or degradation of alpha2 and other regulatory proteins. 3. Molecular genetic characterization of genes identified by mutations causing defects in alpha2 turnover and of genes involved in alpha2 ubiquitination. 4. Molecular genetic analysis of homologous and heterologous short-lived regulatory proteins in yeast to examine mechanistic features of their degradation.